Repeated fed-batch culture methods

ABSTRACT

Provided herein are methods of culturing a microorganism. The method includes providing a container comprising one or more microorganisms and medium, wherein the microorganisms and medium form a start volume. The microorganisms and medium are cultured until the culture reaches a threshold indicator, wherein culturing comprises feeding one or more carbon sources to the culture and wherein the culture is at a threshold volume when the threshold indicator is reached. The method also includes harvesting a portion of the threshold volume to leave a residual volume that is 40% or less of the start volume and adding fresh medium to the container in an amount to return the volume of the culture to the start volume.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/064,694, filed Oct. 16, 2014, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Heterotrophic fermentations of microorganisms including Thraustochytridspecies are efficient ways of generating high value oil and biomassproducts. Under certain cultivation conditions, microorganismssynthesize intracellular oil, which can be extracted and used to producebiofuel (biodiesel, bio-jetfuel, and the like) and nutritional lipids(polyunsaturated fatty acids, e.g. DHA, EPA, DPA). The biomass ofmicroorganisms such as Thraustochytrid species is also of greatnutritional value due to the high PUFA and protein content and can beused as nutritional supplement for animal feed.

Microorganism fermentation processes are carried out mostly in batch orfed-batch processes. Batch processes typically involve a closed systemculture in which cells are grown in a fixed volume of nutrient culturemedium under specific conditions (e.g., specific levels of nutrients,temperature, pressure, and the like) to a certain density in afermenter, harvested and processed as a batch. In typical fed-batchprocesses, one or more nutrients are fed or supplied to a fermenter, inwhich they remain until the end of the culture process. Fed-batchculture processes can be superior to batch culture processes whencontrolling concentrations of a nutrient (or nutrients) affects theyield or activity of a desired product. Oil-producing fermentationprocesses are typically comprised of two cultivation stages, a cellproliferation stage, during which all necessary nutrients are availablefor unlimited culture growth, followed by an oil accumulation stage,during which a key growth nutrient (typically nitrogen) is purposelylimited in the medium while excessive carbon nutrient is provided andchanneled into oil synthesis. When the target cell concentration and oilcontent is reached, the fermentation process is stopped and oil-richbiomass is harvested. The fermenter vessel then must be cleaned,sterilized and re-batched with fresh medium, and a seed train needs tobe ready to inoculate the production vessel again (e.g., a “turnaround”operation between batch/fed-batch fermentations). Such a turnaroundoperation is often time and energy consuming and limits the totalavailable operating hours of the production vessel for an establishedproduction process. Alternatively, microorganisms can be cultured usingcontinuous methods where fresh medium is continuously added to thefermenter, while culture liquid is continuously removed to keep theculture volume constant. Continuous culture processes can be used tomaintain the microorganism at a specific growth rate or physiologicalsteady state but can be difficult to maintain without disruption and aretypically used for research purposes, as fed-batch or batch culturestend to provide better results (e.g., higher oil yield) and are easierto use for large scale production purposes.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods of culturing a microorganism. The methodsinclude providing a container comprising one or more microorganisms andmedium, wherein the microorganisms and medium form a start volume,culturing the microorganisms in the medium until the culture reaches athreshold indicator, wherein culturing comprises feeding one or morecarbon sources to the culture and wherein the culture is at a thresholdvolume when the threshold indicator is reached, harvesting a portion ofthe threshold volume to leave a residual volume that is 40% or less ofthe start volume, and adding fresh medium to the container in an amountto return the volume of the culture to the start volume.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the progression of in-vessel biomassconcentration and oil concentration over time during a repeatedfed-batch fermentation in a 30 L fermenter.

FIG. 2 is a graph showing biomass productivity and oil productivityimprovement throughout a repeated fed-batch fermentation in 30 Lfermenter, as well as constant biomass productivity and oil productivityof fed-batch fermentations. RFB in the legend stands for repeatedfed-batch.

FIG. 3 is a graph showing the progression of in-vessel biomassconcentration and oil concentration over time during a repeatedfed-batch fermentation in a 7 L fermenter.

FIG. 4 is a graph showing biomass productivity and oil productivityimprovement throughout a repeated fed-batch fermentation in 7 Lfermenter, as well as constant biomass productivity and oil productivityof fed-batch fermentations. RFB in the legend stands for repeatedfed-batch.

FIG. 5 is a graph showing the impact of changing residual seed volume(20%, 30%, and 40%) on the overall averaged biomass and oilproductivities. RFB in the axis stands for repeated fed-batch.

DETAILED DESCRIPTION OF THE INVENTION

Methods of cultivating microorganisms and methods of producing oil by arepeated fed-batch process are provided herein. The provided methodsresult in greater overall volumetric productivity of both biomass andoil than a typical batch or fed-batch process. Briefly, the processinvolves cultivating microorganisms in a fed-batch method where, uponcompletion of the fermentation as defined by reaching a particularvolume and/or by meeting volumetric biomass and oil yields, the vesselis drained in a manner which maintains its sterility and leaves behind acertain predetermined volume of culture (e.g., 10% of the initial mediavolume). Fresh, sterile media is then added to the vessel where theculture left behind from the previous fermentation is used as a seed.This process can be repeated indefinitely. The amount of culture leftbehind for use as a seed can vary; however, one should consider thetradeoff between biomass left un-harvested, and the reduced time spentin the lag-phase of the subsequent fermentation. In using a repeatedfed-batch process, fermenter turnaround time is significantly reducedwhich, in turn, leads to higher overall volumetric productivity ofbiomass and oil; far exceeding that of conventional batch and fed-batchprocesses. Also, the repeated fed-batch process minimizes the need forcleaning and sterilization, thereby lowering operating costs.Furthermore, there is less dependence on a seed train, which reducesboth labor and energy costs.

Microorganisms

The methods described herein include extracting lipids from a populationof microorganisms. The population of microorganisms described herein canbe algae (e.g., microalgae), fungi (including yeast), bacteria, orprotists. Optionally, the microorganism includes Thraustochytrids of theorder Thraustochytriales, and, more specifically, Thraustochytriales ofthe genus Thraustochytrium. Optionally, the population of microorganismsincludes Thraustochytriales as described in U.S. Pat. Nos. 5,340,594 and5,340,742, which are incorporated herein by reference in theirentireties. The microorganism can be a Thraustochytrium species, such asthe Thraustochytrium species deposited as ATCC Accession No. PTA-6245(i.e., ONC-T18) as described in U.S. Pat. No. 8,163,515, which isincorporated by reference herein in its entirety. Thus, themicroorganism can have an 18 s rRNA sequence that is at least 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more (e.g., including 100%) identical to SEQ ID NO:1.

The microorganisms for use in the methods described herein can produce avariety of lipid compounds. As used herein, the term lipid includesphospholipids, free fatty acids, esters of fatty acids,triacylglycerols, sterols and sterol esters, carotenoids, xanthophyll(e.g., oxycarotenoids), hydrocarbons, and other lipids known to one ofordinary skill in the art. Optionally, the lipid compounds includeunsaturated lipids. The unsaturated lipids can include polyunsaturatedlipids (i.e., lipids containing at least 2 unsaturated carbon-carbonbonds, e.g., double bonds) or highly unsaturated lipids (i.e., lipidscontaining 4 or more unsaturated carbon-carbon bonds). Examples ofunsaturated lipids include omega-3 and/or omega-6 polyunsaturated fattyacids, such as docosahexaenoic acid (i.e., DHA), eicosapentaenoic acid(i.e., EPA), and other naturally occurring unsaturated, polyunsaturatedand highly unsaturated compounds.

Processes

Provided herein is a method of culturing a microorganism. The methodincludes providing a container comprising one or more microorganisms andmedium, wherein the microorganisms and medium form a start volume;culturing the microorganisms in the medium until the culture reaches athreshold indicator, wherein culturing comprises feeding one or morecarbon sources to the culture and wherein the culture is at a thresholdvolume when the threshold indicator is reached; harvesting a portion ofthe threshold volume to leave a residual volume that is 40% or less ofthe start volume; and adding fresh medium to the container in an amountto return the volume of the culture to the start volume.

The methods are applicable to large-scale fermentation as well assmall-scale fermentation and any fermentation scale between. Large-scalefermentation, as used herein, refers to fermentation in a fermenter thatis at least approximately 1,000 L in volumetric capacity (i.e., workingvolume), leaving adequate room for headspace. Small-scale fermentationrefers generally to fermentation in a fermenter that is generally nomore than approximately 100 L in volumetric capacity, such as 5 L, 10 L,50 L or 100 L. A demonstrated advantage of the present fed-batchfermentation process is that it may be utilized for the production ofoil at the 5-10 L fermenter scale and is scalable to any volume, forexample, 100 L, 150 L, 250 L, 500 L, 1000 L or more, without limitation.

As described in more detail in the examples below, the repeatedfed-batch process alleviates, if not eliminates, the turnaround time ofthe production vessel, with the ultimate goal of increasing volumetricproductivity. An example of how volumetric productivity increases overthat of typical fed batch fermentation is illustrated in FIG. 1.Assuming a 24 hour turnaround time for the production vessel to beincluded in the total process time the overall biomass (X) productivityat any given time can be calculated as: X (gram)/Vessel Working Volume(L)/Time*24 (hours/day) with the final unit being g/L-day. Oilproductivity can be calculated in a similar manner as: Oil (g)/VesselWorking Volume (L)/Time*24 (hours/day). As seen in FIG. 1 biomass andoil productivities of a fed-batch process will remain constant overtime. Conversely, after the first cycle of the repeated fed-batchprocess average productivity increases, far exceeding that of thefed-batch process as turnaround time is not required, and cycle time isdecreased due to increased seed density, in this dataset a 20% seed wasemployed.

In the provided methods, the residual volume can be from 1% to 40% ofthe start volume, e.g., from 1% to 5%, 1% to 10%, 1% to 20%, 1% to 30%,5% to 10%, 5% to 20%, 5% to 30%, 10%, to 20%, 10% to 30%, 20% to 40%, orany volume between 1% and 40% inclusive of the start volume. Optionally,the residual volume is at least about 10% of the start volume.

The provided methods include culturing the microorganisms until theculture reaches a threshold indicator for a parameter. As used herein,the term parameter refers to a variable in the culture conditions whichcan be monitored and controlled to adjust the progress of amicroorganism culture. A threshold indicator is a preselected level orconcentration for a given parameter. Such parameters include, but arenot limited to, volume of the culture, optical density (OD), cellconcentration, carbon dioxide production rate, pH, dissolved oxygen(DO), time, concentration of nutrient in culture medium, accumulation ofmetabolic by-products, temperature, biomass productivity, and oilproductivity. Any suitable parameter or combination of parameters iscontemplated for use as would be understood by a person of ordinaryskill in the art and based upon the guidance provided herein.Optionally, the threshold indicator is a preselected level orconcentration of nutrient(s) in the culture medium. Suitable nutrientsthat can be measured in the culture medium include, but are not limitedto, carbon and nitrogen.

The provided methods optionally include repeating the steps of (i)culturing the microorganisms in the medium until the culture reaches athreshold indicator, wherein culturing comprises feeding one or morecarbon sources to the culture and wherein the culture is at a thresholdvolume when the threshold indicator is reached; (ii) harvesting aportion of the threshold volume to leave a residual volume that is 40%or less of the start volume; and (iii) adding fresh medium to thecontainer in an amount to return the volume of the culture to the startvolume. Optionally, the steps are repeated two or more times.Optionally, the steps are repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.When the process is repeated multiple times, as discussed above, thestart volume and the residual volumes can vary each time or each round.Optionally, the start volumes and residual volumes can remain the sameeach time or each round. By way of example, in a first round, theresidual volume can be 2% of the start volume and in successive rounds,the residual volume can be 10% of the start volume. The residual volumein the successive rounds can also vary, e.g., it can be 10% of the startvolume in one round and 20% of the start volume in another round. Theprovided methods advantageously allow for the culture to be maintainedover a long period of time. As such, the method steps can be repeated aslong as it is desired to maintain the culture and continue to harvest aportion for further use. Optionally, the culture is maintained for aperiod of hours, days, weeks or months. Optionally, the culture ismaintained for at least 150 to 500 hours. For example, the culture canbe maintained for at least 250 hours. Optionally, the culture ismaintained for one, two, three, four, or five weeks.

Optionally, the provided methods include production of a single or onlyone seed or seed train. Typical fed-batch cultivation of microorganismsrequires production of a seed culture produced in a step-wise mannercalled a seed train. The seed train serves to build up the volume anddensity of a culture to inoculate a clean and sterile production vessel.A seed train requires time, energy for sterilization, and also createsmore opportunity for contamination as the culture is transferred betweenmultiple vessels. The repeated fed-batch method requires this seed trainonly to inoculate the first cycle. Likewise, the production vessel onlyneeds to be sterilized for the initial cycle. Therefore, time is savedin turning around the production vessel (cleaning and sterilization) andenergy is saved from cleaning, sterilizing and operating vessels in theseed train. Thus, the provided methods optionally include a singlesterilization step. Moreover, risk of contamination is alleviated fromculture transfers in the seed train for sequential batches. Thus, theprovided methods result in reduced contamination as compared to typicalbatch or fed-batch processes.

Using the production vessel culture (i.e., the residual volume) as theseed for successive batches also allows the choice of selecting thepercentage of seed to use without requiring purchase of larger equipmentor additional fermenters in the seed train. For example, a 2% seedvolume (2000 L for a 100,000 L start volume in a 200,000 L workingvolume production vessel) could be used for the initial batchfermentation, whereas all following iterations could be inoculated witha 10% seed. A 2% seed culture eliminates the need for a larger vessel inthe seed train (i.e., a 10,000 L working volume vessel) alleviatingcapital costs/investment and lowering risk of contamination as there isone less transfer of the seed culture. By using a 2% seed, the lag phaseof microorganism growth is increased, leading to lower volumetricproductivities in the production vessels. However, with successivebatches using the repeated fed-batch method being inoculated with a 10%seed volume this long lag phase is dramatically shortened.

The provided methods include or can be used in conjunction withadditional steps for culturing microorganisms according to methods knownin the art. For example, a Thraustochytrid, e.g., a Thraustochytriumsp., can be cultivated according to methods described in U.S. PatentPublications 2009/0117194 or 2012/0244584, which are herein incorporatedby reference in their entireties for each step of the methods orcomposition used therein.

Microorganisms are grown in a growth medium (also known as “culturemedium”). Any of a variety of medium can be suitable for use inculturing the microorganisms described herein. Optionally, the mediumsupplies various nutritional components, including a carbon source and anitrogen source, for the microorganism. Medium for Thraustochytridculture can include any of a variety of carbon sources. Examples ofcarbon sources include fatty acids, lipids, glycerols, triglycerols,carbohydrates, polyols, amino sugars, and any kind of biomass or wastestream. Fatty acids include, for example, oleic acid. Carbohydratesinclude, but are not limited to, glucose, cellulose, hemicellulose,fructose, dextrose, xylose, lactulose, galactose, maltotriose, maltose,lactose, glycogen, gelatin, starch (corn or wheat), acetate, m-inositol(e.g., derived from corn steep liquor), galacturonic acid (e.g., derivedfrom pectin), L-fucose (e.g., derived from galactose), gentiobiose,glucosamine, alpha-D-glucose-1-phosphate (e.g., derived from glucose),cellobiose, dextrin, alpha-cyclodextrin (e.g., derived from starch), andsucrose (e.g., from molasses). Polyols include, but are not limited to,maltitol, erythritol, and adonitol. Amino sugars include, but are notlimited to, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, andN-acetyl-beta-D-mannosamine. Optionally, the carbon source is glucose.As noted above, in the provided methods, the carbon source is providedat a high concentration, e.g., at least 200 g/L.

Optionally, the microorganisms provided herein are cultivated underconditions that increase biomass and/or production of a compound ofinterest (e.g., oil or total fatty acid (TFA) content).Thraustochytrids, for example, are typically cultured in saline medium.Optionally, Thraustochytrids can be cultured in medium having a saltconcentration from about 0.5 g/L to about 50.0 g/L. Optionally,Thraustochytrids are cultured in medium having a salt concentration fromabout 0.5 g/L to about 35 g/L (e.g., from about 18 g/L to about 35 g/L).Optionally, the Thraustochytrids described herein can be grown in lowsalt conditions. For example, the Thraustochytrids can be cultured in amedium having a salt concentration from about 0.5 g/L to about 20 g/L(e.g., from about 0.5 g/L to about 15 g/L). The culture mediumoptionally includes NaCl. Optionally, the medium includes natural orartificial sea salt and/or artificial seawater.

The culture medium can include non-chloride-containing sodium salts as asource of sodium. Examples of non-chloride sodium salts suitable for usein accordance with the present methods include, but are not limited to,soda ash (a mixture of sodium carbonate and sodium oxide), sodiumcarbonate, sodium bicarbonate, sodium sulfate, and mixtures thereof.See, e.g., U.S. Pat. Nos. 5,340,742 and 6,607,900, the entire contentsof each of which are incorporated by reference herein. A significantportion of the total sodium, for example, can be supplied bynon-chloride salts such that less than about 100%, 75%, 50%, or 25% ofthe total sodium in culture medium is supplied by sodium chloride.

Optionally, the culture medium has chloride concentrations of less thanabout 3 g/L, 500 mg/L, 250 mg/L, or 120 mg/L. For example, culturemedium for use in the provided methods can have chloride concentrationsof between and including about 60 mg/L and 120 mg/L.

Medium for Thraustochytrids culture can include any of a variety ofnitrogen sources. Exemplary nitrogen sources include ammonium solutions(e.g., NH₄ in H₂O), ammonium or amine salts (e.g., (NH₄)₂SO₄, (NH₄)₃PO₄,NH₄NO₃, NH₄OOCH₂CH₃ (NH₄Ac)), peptone, tryptone, yeast extract, maltextract, fish meal, sodium glutamate, soy extract, casamino acids anddistiller grains. Concentrations of nitrogen sources in suitable mediumtypically range between and including about 1 g/L and about 25 g/L.

The medium optionally includes a phosphate, such as potassium phosphateor sodium-phosphate. Inorganic salts and trace nutrients in medium caninclude ammonium sulfate, sodium bicarbonate, sodium orthovanadate,potassium chromate, sodium molybdate, selenous acid, nickel sulfate,copper sulfate, zinc sulfate, cobalt chloride, iron chloride, manganesechloride calcium chloride, and EDTA. Vitamins such as pyridoxinehydrochloride, thiamine hydrochloride, calcium pantothenate,p-aminobenzoic acid, riboflavin, nicotinic acid, biotin, folic acid andvitamin B12 can be included.

The pH of the medium can be adjusted to between and including 3.0 and10.0 using acid or base, where appropriate, and/or using the nitrogensource. Optionally, the medium can be sterilized.

Generally a medium used for culture of a microorganism is a liquidmedium. However, the medium used for culture of a microorganism can be asolid medium. In addition to carbon and nitrogen sources as discussedherein, a solid medium can contain one or more components (e.g., agar oragarose) that provide structural support and/or allow the medium to bein solid form.

Cells can be cultivated over a period of time. Optionally, the cells arecultured for anywhere from 1 day to 60 days. Optionally, the culture ismaintained for a period of hours, days, weeks or months. Optionally, theculture is maintained for at least 150 to 500 hours. Optionally, theculture is maintained for at least 250 hours. Optionally, the culture ismaintained for one, two, three, four, or five weeks. Cultivation isoptionally carried out at temperatures from about 4° C. to about 30° C.,e.g., from about 18° C. to about 28° C. Cultivation can includeaeration-shaking culture, shaking culture, stationary culture, batchculture, semi-continuous culture, continuous culture, rolling batchculture, wave culture, or the like. Cultivation can be performed using aconventional agitation-fermenter, a bubble column fermenter (batch orcontinuous cultures), an airlift fermenter, a wave fermenter, and thelike.

Cultures can be aerated by one or more of a variety of methods,including shaking Optionally, shaking ranges from about 100 rpm to about1000 rpm, e.g., from about 350 rpm to about 600 rpm or from about 100 toabout 450 rpm. Optionally, the cultures are aerated using differentshaking speeds during biomass-producing phases and duringlipid-producing phases. Alternatively or additionally, shaking speedscan vary depending on the type of culture vessel (e.g., shape or size offlask).

The production of desirable lipids can be enhanced by culturing cellsaccording to methods that involve a shift of one or more cultureconditions in order to obtain higher quantities of desirable compounds.Optionally, cells are cultured first under conditions that maximizebiomass, followed by a shift of one or more culture conditions toconditions that favor lipid productivity. Conditions that are shiftedcan include oxygen concentration, C:N ratio, temperature, andcombinations thereof. Optionally, a two-stage culture is performed inwhich a first stage favors biomass production (e.g., using conditions ofhigh oxygen (e.g., generally or relative to the second stage), low C:Nratio, and ambient temperature), followed by a second stage that favorslipid production (e.g., in which oxygen is decreased, C:N ratio isincreased, and temperature is decreased, as compared to the firststage). In contrast to previously described methods, the providedmethods allow for maintaining the culture for a prolonged time underconditions at high levels of oil or lipid production.

Pasteurization

Optionally, the resulting biomass is pasteurized to inactivateundesirable substances present in the biomass. For example, the biomasscan be pasteurized to inactivate compound degrading substances. Thebiomass can be present in the fermentation medium or isolated from thefermentation medium for the pasteurization step. The pasteurization stepcan be performed by heating the biomass and/or fermentation medium to anelevated temperature. For example, the biomass and/or fermentationmedium can be heated to a temperature from about 50° C. to about 95° C.(e.g., from about 55° C. to about 90° C. or from about 65° C. to about80° C.). Optionally, the biomass and/or fermentation medium can beheated from about 30 minutes to about 120 minutes (e.g., from about 45minutes to about 90 minutes, or from about 55 minutes to about 75minutes). The pasteurization can be performed using a suitable heatingmeans, such as, for example, by direct steam injection.

Optionally, no pasteurization step is performed. Stated differently, themethod taught herein optionally lacks a pasteurization step.

Harvesting and Washing

Optionally, the biomass can be harvested according to a variety ofmethods, including those currently known to one skilled in the art. Forexample, the biomass can be collected from the fermentation mediumusing, for example, centrifugation (e.g., with a solid-ejectingcentrifuge) or filtration (e.g., cross-flow filtration). Optionally, theharvesting step includes use of a precipitation agent for theaccelerated collection of cellular biomass (e.g., sodium phosphate orcalcium chloride).

Optionally, the biomass is washed with water. Optionally, the biomasscan be concentrated up to about 20% solids. For example, the biomass canbe concentrated to about 5% to about 20% solids, from about 7.5% toabout 15% solids, or from about solids to about 20% solids, or anypercentage within the recited ranges. Optionally, the biomass can beconcentrated to about 20% solids or less, about 19% solids or less,about 18% solids or less, about 17% solids or less, about 16% solids orless, about 15% solids or less, about 14% solids or less, about 13%solids or less, about 12% solids or less, about 11% solids or less,about 10% solids or less, about 9% solids or less, about 8% solids orless, about 7% solids or less, about 6% solids or less, about 5% solidsor less, about 4% solids or less, about 3% solids or less, about 2%solids or less, or about 1% solids or less.

Isolation and Extraction

The provided methods, optionally, include isolating the polyunsaturatedfatty acids from the biomass or microorganisms. Isolation of thepolyunsaturated fatty acids can be performed using one or more of avariety of methods, including those currently known to one of skill inthe art. For example, methods of isolating polyunsaturated fatty acidsare described in U.S. Pat. No. 8,163,515, which is incorporated byreference herein in its entirety. Optionally, the medium is notsterilized prior to isolation of the polyunsaturated fatty acids.Optionally, sterilization comprises an increase in temperature.Optionally, the polyunsaturated fatty acids produced by themicroorganisms and isolated from the provided methods are medium chainfatty acids. Optionally, the one or more polyunsaturated fatty acids areselected from the group consisting of alpha linolenic acid, arachidonicacid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoicacid, gamma-linolenic acid, linoleic acid, linolenic acid, andcombinations thereof.

Products

Oil including polyunsaturated fatty acids (PUFAs) and other lipidsproduced according to the method described herein can be utilized in anyof a variety of applications exploiting their biological, nutritional,or chemical properties. Thus, the provided methods optionally includeisolating oil from the harvested portion of the threshold volume.Optionally, the oil is used to produce fuel, e.g., biofuel. Optionally,the oil can be used in pharmaceuticals, food supplements, animal feedadditives, cosmetics, and the like. Lipids produced according to themethods described herein can also be used as intermediates in theproduction of other compounds.

By way of example, the oil produced by the microorganisms cultured usingthe provided methods can comprise fatty acids. Optionally, the fattyacids are selected from the group consisting of alpha linolenic acid,arachidonic acid, docosahexaenoic acid, docosapentaenoic acid,eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenicacid, and combinations thereof. Optionally, the oil comprisestriglycerides. Optionally, the oil comprises fatty acids selected fromthe group consisting of palmitic acid (C16:0), myristic acid (C14:0),palmitoleic acid (C16:1(n-7)), cis-vaccenic acid (C18:1(n-7)),docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)),and combinations thereof.

Optionally, the lipids produced according to the methods describedherein can be incorporated into a final product (e.g., a food or feedsupplement, an infant formula, a pharmaceutical, a fuel, etc.) Suitablefood or feed supplements into which the lipids can be incorporatedinclude beverages such as milk, water, sports drinks, energy drinks,teas, and juices; confections such as candies, jellies, and biscuits;fat-containing foods and beverages such as dairy products; processedfood products such as soft rice (or porridge); infant formulae;breakfast cereals; or the like. Optionally, one or more produced lipidscan be incorporated into a dietary supplement, such as, for example, avitamin or multivitamin. Optionally, a lipid produced according to themethod described herein can be included in a dietary supplement andoptionally can be directly incorporated into a component of food or feed(e.g., a food supplement).

Examples of feedstuffs into which lipids produced by the methodsdescribed herein can be incorporated include pet foods such as catfoods; dog foods and the like; feeds for aquarium fish, cultured fish orcrustaceans, etc.; feed for farm-raised animals (including livestock andfish or crustaceans raised in aquaculture). Food or feed material intowhich the lipids produced according to the methods described herein canbe incorporated is preferably palatable to the organism which is theintended recipient. This food or feed material can have any physicalproperties currently known for a food material (e.g., solid, liquid,soft).

Optionally, one or more of the produced compounds (e.g., PUFAs) can beincorporated into a nutraceutical or pharmaceutical. Examples of such anutraceuticals or pharmaceuticals include various types of tablets,capsules, drinkable agents, etc. Optionally, the nutraceutical orpharmaceutical is suitable for topical application. Dosage forms caninclude, for example, capsules, oils, granula, granula subtilae,pulveres, tabellae, pilulae, trochisci, or the like.

The oil or lipids produced according to the methods described herein canbe incorporated into products as described herein in combination withany of a variety of other agents. For instance, such compounds can becombined with one or more binders or fillers, chelating agents,pigments, salts, surfactants, moisturizers, viscosity modifiers,thickeners, emollients, fragrances, preservatives, etc., or anycombination thereof.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

As used throughout, ranges (e.g., 1-10) and references to about a givenvalue (e.g., about 1 or about 10) includes the recited value or values(e.g., 1 and/or 10)

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

The examples below are intended to further illustrate certain aspects ofthe methods and compositions described herein, and are not intended tolimit the scope of the claims.

EXAMPLES Example 1 Repeated Fed-Batch Fermentation for Production ofBiomass and Oil

In the field of microbial oil production, heterotrophic (dark)fermentation is generally considered superior to autotrophic microbialcultivation in terms of process efficiency and product yield. However,it is often hindered by higher fixed capital cost (the cost ofconstructing a vessel-based fermentation plant is generally much higherthan the capital cost of open-pond and raceway type cultivationsystems). Using a repeated fed-batch production process, higher overallvolumetric productivities can be obtained while lowering operatingcosts. This is achieved by minimizing turnaround time of the productionvessel and minimizing energy usage associated with a seed train andsterilization of the production vessel. This means better utilization offixed capital investments (fermenters and associated equipment) andhigher annual production capacity. There is also a reduced capitalinvestment as only an initial seed train is used.

FIG. 1 shows the progression of in-vessel biomass concentration and oilconcentration over time during a repeated fed-batch fermentation in a 30L fermenter. For this experiment, 10% residual volume was employed usingglucose as carbon source. In FIG. 2, a batch to batch turnaround time of12 hours was used to calculate productivities of each independentfed-batch operation, and the same 12 hours turnaround time was used tocalculate the first batch of the repeated fed-batch operation. As seenin FIG. 2, biomass and oil productivities of a typical fed-batch processwill remain constant over time, because each subsequent fed-batchprocess is independently operated from the previous batch with a fixedturnaround time built in-between each fed-batch process. Conversely,after the first cycle of the repeated fed-batch process averageproductivity increases, far exceeding that of the fed-batch process asturnaround time is not required, and cycle time is decreased due toincreased seed density.

FIG. 3 shows the progression of in-vessel biomass concentration and oilconcentration over time during a repeated fed-batch fermentation in a 7L fermenter. For this experiment, 20% residual volume was employed usingglucose as carbon source. In FIG. 4, a batch to batch turnaround time of12 hours was used to calculate productivities of each independentfed-batch operation, and the same 12 hours turnaround time was used tocalculate the first batch of the repeated fed-batch operation. As seenin FIG. 4, biomass and oil productivities of a typical fed-batch processwill remain constant over time, because each subsequent fed-batchprocess is independently operated from previous batch with fixedturnaround time built in-between. Conversely, after the first cycle ofthe repeated fed-batch process average productivity increases, farexceeding that of the fed-batch process as turnaround time is notrequired, and cycle time is decreased due to increased seed density.

Repeated fed-batch fermentations with different residual seed volumes,i.e., 20%, 30%, and 40%, were carried out over a period of 320 hours,each reaching total of six repeated operations. As seen in FIG. 5, allrepeated fed-batch fermentations generated higher overall averagedbiomass and oil productivities when compared to those of singlefed-batch operation. Increasing residual seed volume from 20% to 30%resulted in significant increase in averaged productivities; while afurther increase in residual seed volume from 30% to 40% brought nofurther productivity improvement. This showed the tradeoff betweenbiomass left un-harvested (i.e. used as residual volume for seed), andreduced time spent in the lag-phase of the subsequent fermentation.Under these conditions, the optimum tradeoff point is approximately 30%residual seed volume.

What is claimed is:
 1. A repeated fed batch method of culturing anoil-producing Thraustochytrid microorganism comprising a. a cycle ofsteps comprising: (1) providing a container comprising one or moreoil-producing Thraustochytrid microorganisms and medium, wherein theoil-producing Thraustochytrid microorganisms and medium form a startvolume; (2) culturing the oil-producing Thraustochytrid microorganismsin the medium in the container by a fed-batch method comprising: (a)culturing the oil-producing Thraustochytrid microorganisms underconditions that favor biomass production; and (b) culturing theoil-producing Thraustochytrid microorganisms of step (a) underconditions that favor lipid production until the culture completesfermentation, and reaches a threshold volume when fermentation iscomplete, wherein the threshold volume is greater than the start volumeand wherein the culturing comprises feeding one or more carbon sourcesto the culture; (3) harvesting a portion of the threshold volume of theculture from the container to leave a residual volume in the containerthat is 20% to 40% of the start volume; (4) adding fresh medium to thecontainer in an amount to return the volume of the culture to the startvolume; and b. repeating the steps of the cycle one or more times. 2.The method of claim 1, further comprising detecting during step (2) (b)the volume of the culture, optical density (OD), dissolved oxygen (DO),cell concentration, carbon dioxide production rate, pH, time,concentration of nutrient in culture medium, biomass productivity, oilproductivity, or any combination thereof.
 3. The method of claim 2,wherein the concentration of nutrient in the culture medium is detectedand wherein the nutrient is carbon or nitrogen.
 4. The method of claim2, wherein the volume of the culture is detected.
 5. The method of claim2, wherein the cell concentration is detected.
 6. The method of claim 1,comprising repeating the steps of (2), (3), and (4).
 7. The method ofclaim 6, wherein the steps of the cycle are repeated two or more times.8. The method of claim 6, wherein the steps of the cycle are repeated 2,3, 4, 5, 6, 7, 8, 9, or 10 times.
 9. The method of claim 1, wherein thelipids comprise triglycerides comprising fatty acids selected from thegroup consisting of alpha linolenic acid, arachidonic acid,docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid,gamma-linolenic acid, linoleic acid, linolenic acid, and combinationsthereof.
 10. The method of claim 1, wherein the lipids comprisetriglycerides.
 11. The method of claim 1, wherein the lipids comprisetriglycerides comprising fatty acids selected from the group consistingof palmitic acid (C16:0), myristic acid (C14:0), palmitoleic acid(C16:1(n-7)), cis-vaccenic acid (C18:1(n-7)), docosapentaenoic acid(C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and combinationsthereof.
 12. The method of claim 1, wherein the oil-producingThraustochytrid microorganism is of the family Thraustochytriaceae. 13.The method of claim 12, wherein the oil-producing Thraustochytridmicroorganism is of the genus Thraustochytrium.
 14. The method of claim13, wherein the oil-producing Thraustochytrid microorganism is amicroorganism deposited under ATCC Accession Number PTA-6245.
 15. Themethod of claim 1, wherein the oil productivity is determined.
 16. Themethod of claim 1, wherein the residual volume comprises oil-rich cells.17. A method of producing oil from a culture of oil-producingmicroorganisms comprising a. culturing the oil-producing microorganismsaccording to the method of claim 1, and b. isolating oil from theharvested portion of the threshold volume.
 18. The method of claim 1,wherein the method results in an oil productivity from 40 to 50 g/L/d.19. The method of claim 18, wherein the method results in a biomassproductivity from 50 to 70 g/L/d.
 20. The method of claim 1, wherein oilproductivity increases after each cycle.
 21. The method of claim 1,wherein biomass productivity increases after each cycle.